INTRODUCTION

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THE VERTEBRATE RENAL proximal tubule is responsible for the excretory transport of a large number of potentially toxic chemicals, including waste products of normal metabolism, drugs, environmental pollutants, and drug and pollutant metabolites. These chemicals are handled by several specific, secretory transport systems that remove substrates from the blood and concentrate them in urine (23, 24). As shown in Fig. 1, renal secretion of anionic xenobiotics is a multistep process. Uptake at the basolateral membrane is indirectly coupled to Na through Na--ketoglutarate (-KG) cotransport and -KG/organic anion exchange; within the cell, organic anions are both free to diffuse through cytoplasm and sequestered in mitochondria and vesicles; exit into the urinary space is by carrier-mediated transport and possibly by microtubule and vesicle-dependent processes.

Three recent studies have implicated protein kinase (PK) C in the control of this transport system. These studies show that, in three preparations, nonperfused S2 segment from rabbit kidney (12), opossum kidney (OK) cells in culture (26), and flounder proximal tubule cells in primary culture (9), rates of organic anion transport [p-aminohippurate (PAH) and 2,4-dichlorophenoxyacetate] are altered by phorbol 12-myristate 13-acetate (PMA), a phorbol ester known to activate PKC (21). However, in rabbit tubule segments, phorbol ester increased transport, whereas, in OK cells and flounder proximal tubule cells, PMA decreased transport. The purpose of the present study was to 1) determine how drugs that alter PKC activity affect organic anion (fluorescein, FL) secretion in an intact renal proximal tubule from killifish (Fundulus heteroclitus) and 2) begin to identify the elements of the transport system that are sensitive to PKC.

As discussed previously, renal tissue from teleost fish offers several advantages for the study of secretory transport mechanisms (15, 22). Teleost kidneys contain a high proportion of proximal tubules that are easily isolated and that remain viable for long periods. When tubules are isolated, broken ends rapidly reseal to form a closed, fluid-filled, luminal compartment that communicates only with the medium through the tubular epithelium. Thus this tissue has the appropriate geometry for the study of transepithelial secretion in intact tubules. Moreover, secretory transport mechanisms found in teleost tubules appear to be identical to those found in mammalian proximal tubules (see, e.g., Refs. 17, 18, 22). Finally, when teleost tubules are used along with fluorescent substrates and quantitative fluorescence microscopy, the mechanisms driving both uptake by the cells and secretion into the tubular lumen can be examined (17, 18).

The present results for killifish proximal tubules show that treatments that activate PKC inhibited both FL accumulation in renal cells and uphill FL secretion into the tubular lumen and that PK inhibitors had the opposite effects. Moreover, they show that all of the effects of PKC activators and inhibitors were due to changes in transport at the basolateral membrane of the cells.

	MATERIALS AND METHODS

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Chemicals. FL, PMA, staurosporine, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), carboxyfluorescein diacetate (CFDA), and the CFDA derivative of 3-[1-(3-aminopropyl)-3-indolyl]-4-(1-methyl-3-indolyl)pyrrole-2,5-dione hydrochloride (fim-1 diacetate) were purchased from Molecular Probes (Eugene, OR), and PAH was purchased from Sigma Chemical (St. Louis, MO). All other chemicals were obtained from commercial sources at the highest purity available.

Animals and tissue preparation. Killifish (F. heteroclitus) were collected on Mount Desert Island, ME, and were maintained in tanks with natural, flowing seawater at the Mount Desert Island Biological Laboratory. For some experiments, killifish were collected near Duke University Marine Laboratory (Beaufort, NC) and maintained in tanks with recirculating, artificial seawater at the National Institute of Environmental Health Sciences.

Fish were decapitated, and renal tubular masses were isolated in a marine teleost saline based on that of Forster and Taggart (7) containing (in mM) 140 NaCl, 2.5 KCl, 1.5 CaCl₂, 1.0 MgCl₂, and 20 tris(hydroxymethyl)aminomethane at pH 8.0. All experiments were carried out at 18-20°C. Under a dissecting microscope, each mass was teased with fine forceps to remove adherent hematopoietic tissue. For microscopy, individual killifish proximal tubules were dissected free of the masses and transferred to a foil-covered Teflon chamber (Bionique) containing 1 ml of marine teleost saline with 1-5 µM fluorescent compound or precursor. The chamber floor was a 4 × 4-cm glass coverslip to which the tubules adhered lightly and through which the tissue could be viewed by means of an inverted microscope.

Conventional fluorescence microscopy. The chamber containing renal tubules was mounted on the stage of a Nikon Diaphot inverted microscope fitted with epifluorescence optics, fluorescence objectives (Nikon ×20 phase 3, numerical aperature = 0.8; Olympus ×60 oil, numerical aperature = 1.3), and a 50-W mercury lamp. A Nikon B-1A filter cube was used (460- to 485-nm band-pass excitation filter, 510 nm dichroic filter, 515-nm long-pass emission filter) for FL, CFDA, and fim-1. To avoid photobleaching, a neutral-density filter that passed only 1 or 10% of the excitation light was kept in the light path, and tissue was exposed to light from the mercury lamp for periods of only 1-2 s.

Epifluorescence images were acquired through the microscope side port by use of a Hamamatsu 2400 charge-coupled device (CCD) video camera or a Paultek electronically cooled CCD camera connected to a video card (Scion Video Image LG-3 with 4 Mbytes of onboard memory) in an Apple Macintosh Centris 650 computer. The video card allowed capture and storage of up to eight frames at the video rate (30 frames/s). With the use of image capture and analysis software [National Institutes of Health (NIH) Image version 1.58], incoming images could be displayed at the video rate on a high-resolution computer monitor (Apple), and eight-frame averages were computed and stored on a laser memory optical disk recorder for later analysis.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Organic anion (fluorescein, FL) secretion in renal proximal tubule. Uptake at the basolateral membrane is indirectly coupled to Na through Na--ketoglutarate (-KG) cotransport and FL/-KG exchange. Transport through the cell is by simple diffusion and possibly vesicle-mediated mechanisms. Exit into the tubular lumen is carrier mediated and possibly vesicle and microtubule dependent (for details, see Refs. 23 and 24).

To make a measurement, dye-loaded tubules in the chamber were viewed under reduced, transmitted light illumination. Individual proximal tubules with well-defined lumens and undamaged epithelia were selected, and an epifluorescence image of each was acquired by averaging eight frames. We have shown previously using a similar video microscopy system and glass capillary tubes filled with solutions of known concentrations of fluorescent solutes that the relationship between image fluorescence and dye concentration is approximately linear (18). Calibration of the present system showed a similar linear relationship. However, because of the many uncertainties in relating cellular fluorescence to concentration of fluors in tubules with complex geometry (17, 25), data are reported here as average measured pixel intensity rather than estimated concentration.

Fluorescence intensities were measured from stored images using an Apple Power Macintosh 7100 computer and NIH Image version 1.58 software as described previously (16, 17). Briefly, the fluorescent image of each tubule was magnified electronically 1.5 times, and three to five adjacent cellular and luminal areas (200-400 pixels each) were selected. These were chosen to encompass nearly the entire length of the tubule that was in sharp focus. After background subtraction, the average pixel intensity for each area was calculated, and then the lumen-to-cell fluorescence ratio for each pair of adjacent areas was calculated. The values used for that tubule were the means of all selected areas.

Data are presented as steady-state fluorescence intensity measurements made over the cellular and luminal regions of the tubules as well as paired lumen-to-cell fluorescence ratios. Three caveats must be kept in mind when interpreting such measurements. First, the fluorescence signal from a probe is sensitive to probe environment (pH or solvent polarity). As a result, the relationship between probe fluorescence and concentration could vary with tissue region. Second, in conventional fluorescence microscopy, measurements of cellular fluorescence provide information about the average dye concentration in that compartment. However, because of the light-gathering properties of conventional epifluorescence optics and tubule geometry, luminal fluorescence intensities cannot be measured without including at least some contribution from surrounding cellular layers. This contribution can be minimized greatly by the high numerical aperture objectives used here and by taking images at the plane corresponding to the widest part of the tubule, which keeps the plane of focus for the lumen as far away from cells above and below it. In preliminary experiments, conventional and confocal images of killifish tubules loaded with FL-based dyes were compared. The primary differences were a higher lumen-to-cell fluorescence ratio in confocal images (5-8 with confocal optics vs. 2-4 with conventional optics) and a noticeable loss of cellular detail in the conventional images. When transport was inhibited, similar patterns of effects were seen with both techniques, that is, inhibition of basolateral uptake reduced cellular and luminal fluorescence roughly in parallel, and inhibition of efflux into the lumen primarily reduced luminal fluorescence (unpublished data and Ref. 17). Finally, a change in steady-state solute distribution between tissue compartments merely indicates that one or more processes have been altered. Additional information is needed to identify the affected mechanism.

Confocal fluorescence microscopy. Tubules in a Bionique chamber were mounted on the stage of a Zeiss model 410 inverted laser scanning confocal microscope and were viewed through a Zeiss ×10 plan-neofluar objective. To collect fluorescent images, tubules were illuminated by an Ar-Kr laser at 488 nm. A 510-nm dichroic filter was positioned in the light path, and a 515-nm long-pass emission filter was placed in front of the detector. Each image was a single 8-s scan. To minimize photobleaching, images were collected at 20% laser power, with neutral density filters passing only 1-3% of the light. Preliminary experiments showed that, under these conditions, photobleaching was minimal, that is, fluorescence intensities in cells and tubular lumens were reduced by <5% in consecutive 8-s scans. Confocal images (512 × 512 × 8 bits) were viewed on a high-resolution monitor and were saved to an optical disk for transfer to the Power Macintosh computer for viewing and analysis using NIH Image software.

Statistics. Data are given as means ± SE. Means were considered to be statistically different at P < 0.05 by use of the appropriate paired or unpaired t-test.

	RESULTS

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Organic anion transport. Epifluorescence micrographs of control tubules incubated in FL-containing media exhibit the same steady-state fluorescence distribution pattern seen previously (17-19), that is, fluorescence intensity in the lumen is greater than that in the cell, which is greater than that in the bath (Fig. 2). This pattern is also identical to that seen using colored organic anions (13) and has been confirmed using confocal microscopy (Refs. 14, 17, 19, and the present study). It is interpreted to demonstrate two steps in organic anion secretion in these tubules, one step at each face of the epithelium. A similar fluorescence distribution pattern was found for tubules exposed to the phorbol ester PMA. However, when compared with controls, PMA-exposed tubules exhibited a concentration-dependent reduction in cellular and luminal fluorescence (Fig. 2). Figure 3A shows the effects of 1-100 nM PMA on the steady-state distribution of FL fluorescence obtained by quantitative image analysis (conventional optics). At all concentrations studied, the phorbol ester significantly reduced both cellular and luminal fluorescence, with the magnitude of the effect increasing with increasing PMA concentration. For example, 1 nM PMA reduced cellular and luminal fluorescence by 21 and 37%, respectively, whereas 100 nM PMA reduced cellular and luminal fluorescence by 55 and 64%, respectively (Fig. 3A). Note that lumen-to-cell fluorescence ratios were essentially unchanged by PMA (Fig. 3B). Similar results were found when a PMA dose response was obtained using confocal optics (Fig. 3C). Again, both cellular and luminal fluorescence fell with increasing PMA. A plot of cellular vs. luminal fluorescence for all tubules (control and PMA exposed) was well described by a simple linear regression (Fig. 3D), indicating that PMA reduced fluorescence in both regions of the tubules in parallel.

View larger version (138K): [in this window] [in a new window]   Fig. 2.   Fluorescence micrographs (conventional optics) of killifish renal proximal tubules after 30-min incubation in medium with 1 µM FL and 0 (A), 1 (B), 10 (C), and 100 (D) nM phorbol 12-myristate 13-acetate (PMA). Notice the progressive fall in cellular and luminal fluorescence with increasing PMA concentration.

View larger version (28K): [in this window] [in a new window]   Fig. 3.   PMA inhibition of FL transport in killifish tubules. Tubules were incubated for 30 min in medium with 1 µM FL and 0 (control) to 100 nM PMA. Results are for images collected using conventional (A and B) and confocal (C and D) optics. Data are given as means ± SE for 6-10 tubules. Statistical comparisons: * significantly lower than controls, P < 0.05; ** significantly lower than controls, P < 0.01. Broken line in D is the regression line for tubules from all treatments (slope = 4.3; r = 0.97, P < 0.01).

If PMA was acting through a PK to inhibit FL transport, then its inhibitory effects should be blocked by PK inhibitors. Figure 4 shows the results of an experiment in which tubules were incubated in medium without (control) or with 100 nM PMA or 100 nM PMA plus 1 µM staurosporine. PMA reduced cellular and luminal fluorescence by ~50%, but no such reduction was found for tubules exposed to PMA plus staurosporine.

View larger version (11K): [in this window] [in a new window]   Fig. 4.   Reversal of PMA inhibition of FL transport by staurosporine. Tubules were incubated for 30 min in medium with 1 µM FL and no additions (control), 100 nM PMA or 100 nM PMA plus 1 µM staurosporine (Stauro). Images acquired using conventional optics. Data are given as means ± SE for 7-11 tubules. Statistical comparisons: ** significantly lower than controls, P < 0.01.

The time course of 100 nM PMA action on FL accumulation in killifish tubules is shown in Fig. 5. In agreement with previous results demonstrating that control tubules rapidly reach steady state with regard to FL fluorescence (19), the control tubules exhibited roughly constant fluorescence in both lumen and cells from 15 min onward. The PMA-treated tubules also showed roughly constant fluorescence over time in both regions of the tissue, but values were substantially below controls. Because both cellular and luminal fluorescence were reduced by PMA, the lumen-to-cell fluorescence ratio was only slightly changed. Thus PMA inhibition of FL uptake and secretion was rapid, occurring within 15 min.

View larger version (12K): [in this window] [in a new window]   Fig. 5.   Time course of PMA inhibition of FL transport in killifish tubules. Tubules were incubated for the time indicated in medium with 1 µM FL and 0 (control) or 100 nM PMA. Images were acquired using conventional optics. Data are given as means ± SE for 8-12 tubules. Statistical comparisons: ** significantly lower than controls, P < 0.01.

If an activator of PKC inhibited FL transport, would kinase inhibitors stimulate? To answer this question, tubules were exposed to two PK inhibitors, staurosporine and H-7, during 30-min FL transport experiments. Both PK inhibitors significantly increased luminal and cellular fluorescence (Fig. 6). For both, greater effects were generally found with higher concentrations. Additional experiments were carried out with staurosporine. It had near maximal effects on FL transport within 10 min (data not shown). As demonstrated in Fig. 7, the increases in cellular and luminal fluorescence caused by staurosporine were blocked by the model substrate for the organic anion transport system, PAH. Thus FL uptake into cells and secretion into the tubular lumen were increased by PK inhibitors, and those increases appeared to be mediated by the organic anion transport system.

View larger version (32K): [in this window] [in a new window]   Fig. 6.   Stimulation of 1 µM FL transport in killifish tubules by protein kinase inhibitors. Tubules were incubated for 30 min in medium with 1 µM FL and no additions (control) or the indicated concentration of staurosporine (A and B) or H-7 (C and D). Images were acquired using conventional optics. Data are given as means ± SE for 10-11 tubules. Statistical comparisons: * significantly higher than controls, P < 0.05; ** significantly higher than controls, P < 0.01.

View larger version (12K): [in this window] [in a new window]   Fig. 7.   p-Aminohippurate (PAH) blocks staurosporine-induced stimulation of FL transport. Tubules were incubated for 30 min in medium with 1 µM FL and no additions (control), 1 mM PAH, 1 µM staurosporine, or 1 µM staurosporine plus 1 mM PAH. Images were acquired using conventional optics. Data given as means ± SE for 7-12 tubules. Statistical comparisons: ** significantly different from controls, P < 0.01.

Organic anion secretion in proximal tubules is driven by mediated processes at both the luminal and basolateral membranes of the epithelial cells (Fig. 1). As a result of this model of transporters in series, one would expect inhibitors that act at the basolateral membrane to directly reduce uptake into the cells but also to indirectly reduce transport into the lumen. Thus most of the changes seen with PKC effectors could have resulted from altered transport at the basolateral membrane alone or from altered transport at both the basolateral and luminal membranes. To distinguish between these possibilities, imaging studies were carried out using a nonfluorescent substrate (CFDA) that enters cells by simple diffusion and is then converted to a fluorescent organic anion (carboxyfluorescein, CF) intracellularly (17). Conventional and confocal images of killifish proximal tubules exposed to CFDA show that CF accumulates within cells and is secreted into the lumen. In these experiments, accumulation of CF by the cells was only slightly reduced by the organic anions (PAH and probenecid), but both organic anions blocked transport into the lumen (17). Although the latter result is consistent with transport of CF into the lumen by a process that is shared with PAH and probenecid, the absence of a substantial effect of these organic anions on the steady-state cellular accumulation of CF suggests that cellular CF levels are not largely affected by changes in organic anion transporter function (see DISCUSSION).

Figure 8 shows that, when control killifish tubules were incubated in medium with 5 µM CFDA, luminal fluorescence exceeded cellular fluorescence by a factor of two to three. As demonstrated previously (17), addition of 1 mM PAH to the medium had, at most, a small effect on cellular fluorescence and a much larger effect on luminal fluorescence (Fig. 8). Figure 8 also shows that, in contrast to PAH, neither PMA nor staurosporine had any consistent effects on cellular or luminal fluorescence. Thus PKC effectors did not alter CF transport from cell to lumen or CF accumulation within renal cells.

View larger version (19K): [in this window] [in a new window]   Fig. 8.   Lack of effect of PMA and staurosporine on transport of carboxyfluorescein (from carboxyfluorescein diacetate, CFDA) in killifish tubules. Tubules were incubated for 30 min (A) or 60 min (B) in medium with 1 µM CFDA and no additions (control) or with 1 mM PAH, 100 nM PMA, or 1 µM staurosporine. Images were acquired using conventional optics. Data are given as means ± SE for 10-11 tubules. PAH significantly reduced luminal fluorescence (P < 0.01).

Activation of renal PKC by PMA. Cell fractionation and immunocytochemical studies have shown that one indicator of activation of PKC by phorbol ester is migration of enzyme from the cytoplasm to the plasma membrane (21). To determine whether phorbol ester triggered migration of PKC in killifish proximal tubules, I measured the distribution of a high-affinity, fluorescent PKC inhibitor (fim-1) in control and PMA-exposed tubules. Fim-1, like other bisindolylmaleimide inhibitors of PKC, has been shown bind to the ATP site of PKC with high affinity and to exhibit a PKC-to-PKA selectivity ratio of at least 10 (6). In one study, fim-1 was used to examine PMA-induced PKC migration in a rat embryo fibroblast cell line that overexpressed the PKC isoform, PKC1. In these cells, staining patterns for fim-1 and an antibody to PKC1 were similar, with the major difference being some additional mitochondrial staining with fim-1 (6). Although the experiments were conducted with cells that had been treated with PMA and then fixed, the authors suggested the possibility that membrane-permeant fluorescent bisindolylmaleimide derivatives might be used to study PKC distribution in vivo (6).

In the present study, two procedures were used to localize PKC in killifish tubules. First, control and PMA-exposed tubules were fixed and permeabilized and then incubated with fim-1. Second, live control and PMA-exposed tubules were incubated in medium containing a membrane-permeant form of fim-1 [fim-1 diacetate (6)] that is rapidly taken up by cells and then hydrolyzed to fim-1 by intracellular esterases. Initial experiments showed that when live control tubules were incubated in medium with fim-1 or fim-1 diacetate, dye was rapidly secreted into the lumen. In both cases, secretion was blocked by PAH and probenecid. In subsequent experiments with live tubules, 1 mM PAH was added to the medium to prevent secretion of fim-1 into the lumen.

Fim-1 staining of both fixed and live tubules resulted in the same basic inhibitor distribution patterns. Representative conventional and confocal micrographs of fim-1-stained, living tubules are shown in Fig. 9. In control tubules, fluorescence was distributed diffusely through the cells, with some areas of punctate fluorescence being evident. In tubules exposed to 50-200 nM PMA for 15-30 min, diffuse fluorescence was also seen, but now fluorescence was more concentrated at the plasma membrane. This was most evident at the luminal pole of the epithelial cells, but some areas of concentrated staining at the basolateral membrane were also seen. These changes in fluorescence distribution pattern are best seen in low-magnification confocal images (Fig. 9, C and D). Note that the confocal micrographs clearly show that the lumen of the tubule did not accumulate fim-1, rather, the dye was localized to the luminal membrane. Thus, exposure of tubules to PMA caused a fluorescent probe for PKC to migrate from the cytoplasm to the plasma membrane. Both the basolateral and luminal membranes were labeled, but labeling was much more intense along the brush-border membrane.

View larger version (164K): [in this window] [in a new window]   Fig. 9.   PMA treatment causes redistribution of fluorescent PKC inhibitor (fim-1) in killifish tubules. Shown are fluorescence micrographs of control (A and C) and PMA-treated (B and D) tubules. A and B show conventional epifluorescence images; C and D show confocal imges. Tubules were first loaded in medium with 2 µM fim-1 diacetate plus 1 mM PAH. After 1 h, tubules were transferred to medium without (control) and with 100 nM PMA. After 20 min, tubules were placed in a chamber, and images were collected.

	DISCUSSION

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The PKC family of enzymes was defined originally as group of serine/threonine kinases that are activated by calcium, phospholipid, and diacylglycerol (20, 21). PKC isoforms have been implicated in the control of many essential cellular processes, including cell division, metabolism, and membrane transport. In renal proximal tubule cells, PKC affects transport of ions and nutrients by altering processes at both the basolateral and luminal membranes (see, e.g., Refs. 1 and 8). Recent studies have shown that pharmacological agents that alter PKC activity also affect organic anion transport in renal proximal tubules. Hohage et al. (12) measured PAH accumulation by the cells of nonperfused S2 segments of rabbit proximal tubules and found stimulation with PMA and diacylglycerol and inhibition with staurosporine. Very different results were reported in two studies of organic anion transport in cultured renal cells from opossum (26) and from flounder (9), two preparations that secrete organic anions. These latter studies showed inhibition of transepithelial organic anion secretion by PMA and diacylglycerol. In all three studies, PMA effects were blocked by PK inhibitors.

The present imaging study shows that exposure of intact killifish renal proximal tubules to drugs that affect PKC rapidly alters their ability to transport the fluorescent organic anion FL. The phorbol ester PMA, at nanomolar concentrations, partially reduced FL fluorescence in both cells and tubular lumen. PMA effects were blocked by the PK inhibitor staurosporine. Staurosporine and H-7 increased both cellular and luminal fluorescence, and those increases were blocked by PAH, indicating that they represented augmented transport on the organic anion system. From these data for killifish tubules, it would appear that treatments that activated PKC reduced FL uptake and secretion and that treatments that inhibited PKC stimulated FL uptake and secretion. Clearly, in the following two important respects, the present results from intact, actively secreting proximal tubules confirm the findings of Takano et al. (26) and Halpin and Renfro (9): inhibition of secretory transport by PMA and reversal of PMA inhibition by staurosporine. It is not clear why the present results and those of Takano et al. (26) and Halpin and Renfro (9) differ so dramatically from those of Hohage et al. (12), but species differences in responses to pharmacological agents or in cellular signaling mechanisms are certainly possibilities.

The present results provide new information about the role of PKC in the regulation of renal organic anion transport. They show that PKC controls organic anion transport in an intact, secreting proximal tubule. Moreover, not only did PMA inhibit FL transport, but PK inhibitors significantly stimulated FL transport on the organic anion system. Stimulation of transport by PK inhibitors has not been demonstrated previously (9, 12, 26). Thus it appears that, in freshly isolated killifish tubules, the regulatory set point for organic anion transport is near the middle of the range of function, that is, transport can be both increased and decreased by altering PKC activity.

The present data also indicate which element of the organic anion transport system is affected by changes in PKC activity. Secretion of organic anions from blood to urinary space is a multistep process involving uptake at the basolateral membrane of renal cells, movement across the cell interior, and efflux across the luminal membrane into the tubular lumen (Fig. 1). Two findings implicate the first step in secretion, uptake at the basolateral membrane, as the one affected by PKC activation and inhibition. First, with FL as substrate, PMA, staurosporine, and H-7 altered both cellular and luminal fluorescence in parallel. As a result, lumen-to-cell fluorescence ratios were essentially unchanged, even though PMA reduced regional fluorescence intensities by more than one-half and staurosporine increased regional fluorescence intensities by nearly a factor of two. Also, confocal images of FL transport in killifish tubules show that both cellular and luminal fluorescence decreased with PMA (Fig. 3, C and D) and increased with staurosporine (unpublished data). Thus similar effects of PKC modifiers were found in images obtained with confocal and conventional optics. This is consistent with out-of-focus cellular fluorescence making only a small contribution to measured luminal fluorescence with the conventional optics used here (see MATERIALS AND METHODS for a more detailed discussion of this point). Note that, because the basolateral and luminal membranes are arranged in series (Fig. 1), one expects to see changes in both cellular and luminal fluorescence when the basolateral step in organic anion secretion is modified by treatments that alter forces driving basolateral uptake (glutarate, lithium, and ouabain) or that compete with FL for access to the basolateral transporter (PAH and probenecid; see Refs. 14 and 17-19).

The second type of experiment suggesting that the basolateral step in FL transport is the one affected by PKC utilized a nonfluorescent substrate (CFDA) that was taken up by simple diffusion and generated a fluorescent organic anion (CF) intracellularly (17). In these experiments (CFDA added to the medium), PAH and probenecid blocked transport of CF from cell to lumen (Fig. 8 and Ref. 17), indicating that this step in CF transport is mediated by a process that is shared with PAH and probenecid. In contrast, our understanding of the factors that determine steady-state cellular CF levels is incomplete. Preliminary confocal imaging studies of CF transport in killifish tubules (CF added to the medium) have shown that uptake at the basolateral membrane is inhibited by PAH and ouabain and stimulated by low concentrations of glutarate (unpublished data). Thus CF does appear to be a substrate for the same basolateral, Na-dependent uptake system that transports PAH and FL. Yet, experiments in which tubules were incubated in medium with CFDA show that PAH and probenecid have only small effects on steady-state cellular CF levels (Ref. 17 and present study). These results suggest that, in CFDA experiments, steady-state cellular levels of CF must be determined primarily by processes other than those that drive organic anion transport. These might include enzymatic and thermal conversion of CFDA to CF and diffusional uptake and efflux of CFDA. Additional experiments are needed to more completely understand the factors that determine cellular CF levels.

Irrespective of the mechanisms that determine cellular CF levels, the present experiments with CFDA show that neither PMA nor staurosporine had any effects on cellular or luminal fluorescence. Because CF is both compartmentalized intracellularly and secreted into the tubular lumen on the organic anion system (17), the lack of PMA and staurosporine effects indicates that neither organic anion transport across the luminal membrane nor intracellular sequestration of organic anions is regulated by PKC. These findings are not inconsistent with the data of Halpin and Renfro (9), which suggest that dopaminergic inhibition of organic anion secretion by monolayers of flounder proximal tubule cells is achieved through PKC inhibition of Na-K-ATPase. Organic anion uptake at the basolateral membrane is driven by indirect coupling to Na through Na-divalent organic anion cotransport and divalent organic anion-monovalent organic anion exchange (23, 24). Inhibition of Na-K-ATPase would be expected to reduce the magnitude of the Na gradient that indirectly provides the potential energy for organic anion uptake. This in turn would reduce organic anion uptake and subsequently the ability of renal cells to deliver organic anions to the lumen. It remains to be determined whether other processes that directly or indirectly influence organic anion uptake at the basolateral membrane, for example, mitochondrial -KG production, Na--KG cotransport, or FL/-KG exchange, are also affected by changes in PKC activity.

Postulating a basolateral target for PKC action would appear to contradict the results of experiments with fim-1, a fluorescent PKC inhibitor. These experiments show that, as seen in other cell types (21), PMA induced cytoplasm-to-plasma membrane translocation of PKC in killifish tubules. However, in PMA-exposed tubules, fim-1 labeling was most intense along the luminal membrane and was barely detectable along the basolateral membrane. For killifish tubules, we do not know the relative affinities of fim-1 for the various PKC isoforms, nor do we know which isoforms are expressed and in what amounts. Thus one possibility is that the isoform(s) responsible for signaling the reduction in basolateral organic anion uptake might only represent a small fraction of total fim-1-reactive and PMA-sensitive PKC isoforms. Alternatively, PKC control of organic anion transport might be indirect, involving initial phosphorylation of a target protein in the cytoplasm or on the luminal membrane followed by diffusion of the protein or of the signal to some element of the basolateral organic anion transport machinery.

In laboratory animals, renal organic anion (PAH) excretion increases early in life and falls with senescence. In large part, these changes occur independently of altered hemodynamics, since parallel effects are seen in studies with renal cortical slices in which changes in the apparent Michaelis constant and maximal velocity for PAH uptake have been reported (2, 10, 11, 27). In addition, studies of PAH excretion in vivo and of PAH accumulation by renal cortical slices in vitro show that alterations in the hormonal status of the animal are accompanied by changes in transport. For example, dosing rats with thyroid hormones, glucocorticoids, and testosterone increases transport, and castration and dosing with dopamine derivatives and reserpine decreases transport; in every case, responses are age dependent (2-5). Although these and other studies certainly suggest that the renal organic anion transport system is physiologically regulated, it is not yet clear which extracellular signals (hormones, endogenous substrates, and xenobiotics) initiate changes in transport function or how recent studies implicating PKC in the control of transport at the cellular level are related to regulation in vivo. Further studies are needed to identify the specific extracellular signals that signal altered transport, specific links in the intracellular signaling chain (PKC isoforms, metabolites from other signaling systems), and immediate targets of intracellular messengers (Na-K-ATPase, organic anion transporters, cytoskeleton).